CN114107259B - Application of alkaline thermophilic rhamnosidase in preparation of active bioflavonoids - Google Patents

Application of alkaline thermophilic rhamnosidase in preparation of active bioflavonoids Download PDF

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CN114107259B
CN114107259B CN202111511848.7A CN202111511848A CN114107259B CN 114107259 B CN114107259 B CN 114107259B CN 202111511848 A CN202111511848 A CN 202111511848A CN 114107259 B CN114107259 B CN 114107259B
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rhamnosidase
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leu
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CN114107259A (en
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赵国刚
李安安
龙雪
李仲凯
刘铁汉
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Beijing Tuowei Biotechnology Co ltd
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Abstract

The invention relates to an alkaline thermophilic rhamnosidase, which has high conversion rate of flavonoid compounds by using the alkaline thermophilic rhamnosidase under high temperature conditions of 60-100 ℃ and different pH conditions of 5-11, wherein the conversion rate of the flavonoid compounds can reach more than 98.5%.

Description

Application of alkaline thermophilic rhamnosidase in preparation of active bioflavonoids
Technical Field
The invention relates to the field of biology, in particular to application of alkaline thermophilic rhamnosidase in preparation of active bioflavonoids.
Background
alpha-L-rhamnosidase (EC3.2.1.40) is a hydrolase that specifically hydrolyzes the alpha-1, 2, alpha-1, 3, alpha-1, 4, alpha-1, 6 glycosidic bond of a glycan or glycoside compound terminal, releasing L-rhamnose, yielding a new glycan or glycoside compound. alpha-L-rhamnosidase is widely available in nature and can be isolated from bacterial, yeast, fungal, plant and animal tissues. It has been reported that most of the α -L-rhamnosidases are derived from microorganisms, of which 19 have been characterized as bacterial sources of GH78 family α -L-rhamnosidases, and most are derived from gram-positive bacteria including bacillus, lactobacillus, clostridium, streptomyces and thermomycetoma. The CAZY database classifies glycoside hydrolases according to amino acid sequence similarity, and the microbial sources of α -L-rhamnosidase are comprised in the GH13, GH28, GH78 and GH106 glycoside hydrolase families.
The alpha-L-rhamnosidase has good development prospect and application value in the aspects of food production, medical processing, chemical industry and the like. As a biocatalyst, the alpha-L-rhamnosidase can be used for debittering of fruit juice by hydrolyzing naringin in citrus fruit juice, enhancing the aroma of wine by hydrolyzing terpene glycoside, clarifying the fruit juice by hydrolyzing hesperidin, improving the bioavailability and biological activity of flavonoid compounds by hydrolyzing rutin, and can be synthesized into alpha-L-rhamnoside by reverse hydrolysis of the alpha-L-rhamnosidase. The alpha-L-rhamnosidase can convert saponins compounds in common Chinese medicinal materials such as ginsenoside, saikosaponin C and dioscin into active components. Can also be used for specifically converting naringin into highly active prunin. In addition, α -L-rhamnosidase can hydrolyze α -L-rhamnosyl groups at the ends of many natural medicinal ingredients, the hydrolysis product is a key precursor for the synthesis of bioactive isoquercetin (enzymatically modified isoquercitrin, EMIQ) and has been approved as a variety of food additives.
Flavonoids are a class of natural products of polyphenols with a 2-phenylchromone structure that are widely distributed in plants. Studies have shown that: the flavonoids have various physiological and pharmacological effects including antioxidant effect, blood sugar and blood pressure lowering, antiinflammatory, antitumor effect, antibacterial and antiviral effect, cardiovascular disease treatment, intestinal flora regulation, etc. The natural flavonoids are usually stable in the form of glycosides. The flavonoid often contains one or more rhamnosyl groups (including 3-position and 7-position of flavone), and rutin, naringin, hesperidin, neohesperidin, epimedin C and the like are common. In this series of flavone compounds, the presence of rhamnosyl affects their solubility and biological activity. The removal of rhamnoside can obviously improve the bioavailability of natural flavone and exert the biological efficacy to the greatest extent. The enzymatic hydrolysis has the characteristics of mildness, high efficiency, specificity and the like, and is suitable for the hydrolysis of rhamnoside flavonoid compounds, so that the flavonoid rhamnoside hydrolase with high catalytic activity has great application potential.
However, most of flavonoids are used as substrates, and have poor water-solubility and fat-solubility, and are only dissolved in hot dilute alkali solution or certain organic cosolvents (such as methanol), so that the industrial application of rhamnosidase is greatly limited. The current enzymatic conversion and optimal conditions of rhamnosidase are as the bacterial-source alpha-L-rhamnosidase described in patent application CN202110690007.0, the optimal pH is 7, the optimal temperature is 50 ℃, the conversion reaction of patent application CN201910435031.2 is carried out at pH6.0 and 37 ℃, the conversion reaction of patent application CN201810110773.3 is carried out at the temperature of 40-60 ℃ and the pH of 5.0-7.5, the optimal temperature of CN201610635941.1 is 60 ℃, the optimal pH is 5.0, and the enzymatic conversion of flavonoid substances under the higher temperature and more alkaline environment is difficult to realize in the prior art. Therefore, in order to make up the defects of the prior art, break through the limit of the solubility of flavonoid substances, a rhamnosidase which is stable in high temperature resistance and can have high activity under alkaline conditions is needed to be beneficial to the conversion of the flavonoid substances.
Disclosure of Invention
The invention develops a method for carrying out enzymatic conversion of flavonoid compounds under high temperature conditions and different pH conditions, wherein the high temperature conditions are 60-100 ℃, preferably 80 ℃; the different pH conditions are pH5-11, preferably alkaline conditions are pH8-10, and the conversion rate of the flavonoid compounds can reach more than 98.5%.
The alkaline thermophilic rhamnosidase provided by the invention has the optimal temperature of 100 ℃ and the optimal pH of 10, has high temperature stability and pH stability, has high enzyme activity and high efficiency of converting flavonoid substances, does not need other cosolvent in the conversion process, and does not find that other alpha-L-rhamnosidase can reach the level at present.
The object of the present invention is therefore a method for bioconversion of flavonoids with high conversion rates, characterized in that the bioconversion is carried out under conditions of high temperature and different pH, under the action of alkaline thermorhamnosidase. The amino acid sequence of the alkaline thermophilic rhamnosidase is shown in SEQ ID NO: 1.
The flavonoid is one or more selected from rutin, naringin, hesperidin, neohesperidin and epimedin C.
The high temperature condition is 60-100 ℃, preferably 80 ℃; the different pH conditions are pH5-11, preferably alkaline conditions of pH8-10.
Another object of the present invention is to provide an alkaline thermophilic rhamnosidase, which is characterized in that the amino acid sequence of the alkaline thermophilic rhamnosidase is as shown in SEQ ID NO: 1. The gene of the alkaline thermophilic rhamnosidase codes for 894 amino acids, and the theoretical molecular weight of the alkaline thermophilic rhamnosidase is 101.1kDa.
It is another object of the present invention to provide a recombinant vector or host cell containing the gene encoding alkaline thermophilic rhamnosidase.
Another object of the present invention is to provide a method for preparing the alkaline thermophilic rhamnosidase, characterized by comprising the steps of:
1) Transforming the host cell with the recombinant vector to obtain a recombinant strain;
2) Culturing a recombinant strain, inducing the alkaline thermophilic rhamnosidase expression;
3) Recovering and purifying the expressed alkaline thermophilic rhamnosidase.
It is a further object of the present invention to provide the use of said alkaline thermophilic rhamnosidase in bioconversion of flavonoids under high temperature conditions and/or alkaline conditions. The flavonoid is one or more selected from rutin, naringin, hesperidin, neohesperidin and epimedin C. The high temperature condition is 60-100 ℃, preferably 80 ℃; the different pH conditions are pH5-11, preferably alkaline conditions of pH8-10.
The invention aims to overcome the defects of the prior art and provide the novel alkaline thermophilic rhamnosidase which has excellent properties and is suitable for being applied to foods and medicines. The alkaline thermophilic rhamnosidase has the following advantages:
1. the optimum pH of the rhamnosidase of the invention is 10.0, and the rhamnosidase has 90% activity at pH 5.0-10.0; when the enzyme is mixed with buffer solution with pH of 5-10 and incubated for 2 hours, the activity of more than 95% can be maintained, and the pH stability is good. The pH condition for conversion is pH5-11, preferably alkaline condition pH8-10.
2. The optimum temperature of the rhamnosidase is 100 ℃, the temperature stability is high, and 100% of activity can be maintained after incubation for 2 hours at 60-80 ℃. The conversion temperature is 60-100 ℃, preferably 80 ℃.
3. The rhamnosidase of the invention has high enzyme activity, and the highest enzyme activity of the rhamnosidase which has been researched in the prior literature is the rhamnosidase from thermophilic bacterium, which reaches 352 enzyme activity units; the enzyme activity of the rhamnosidase of the invention can reach 2974 enzyme activity units, which is far higher than the enzyme activity of various known rhamnosidase.
Drawings
FIG. 1 is a diagram showing hydrolysis lines of substrates related to rhamnosidase hydrolysis according to the present invention.
FIG. 2 is a SDS-PAGE analysis of recombinant rhamnosidase expressed in E.coli, wherein M: protein markers; 1: crude enzyme solution; 2: purified recombinant rhamnosidase.
FIG. 3 is the optimal pH of the recombinant rhamnosidase.
FIG. 4 is the pH stability of recombinant rhamnosidase.
FIG. 5 shows the optimum temperature of recombinant rhamnosidase.
FIG. 6 is the thermostability of recombinant rhamnosidase.
FIG. 7 is an HPLC detection of the hydrolytic conversion of rutin by recombinant rhamnosidase.
FIG. 8 is an HPLC assay for the hydrolytic conversion of naringin by recombinant rhamnosidase.
FIG. 9 is a HPLC detection of the hydrolytic conversion of recombinant rhamnosidase into hesperidin.
FIG. 10 is an HPLC detection of epimedin C converted to epimedium by hydrolysis of recombinant rhamnosidase.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example one Experimental reagent and related conditions
Related reagents
isopropyl-beta-D-thiogalactoside (IPTG), kanamycin sulfate, agar powder (Solarbio company), ni-NTA Resin (Beijing full gold biotechnology Co., ltd.), and protein gel kit (Kangji Biotech Co., ltd.)
Related solutions:
liquid LB medium: 5g of yeast extract, 10g of tryptone and 10g of NaCl, and distilled water is dissolved to a volume of 1L.
Solid LB medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl, distilled water after dissolution to a volume of 1L, and 15g of agar powder.
Ni-NTA Lysis buffer：10mM Imidazole，300mM NaCl，50mM NaH 2 PO 4 After dissolution, the pH was adjusted to 8.0 and distilled water was set to 1L.
Ni-NTA Washing buffer：50mM Imidazole，300mM NaCl，50mM NaH 2 PO 4 After dissolution, the pH was adjusted to 8.0 and distilled water was set to 1L.
Ni-NTA 250mM Elution buffer：250mM Imidazole，300mM NaCl，50mM NaH 2 PO 4 After dissolution, the pH was adjusted to 8.0 and distilled water was set to 1L.
Ni-NTA350mM Elution buffer：350mM Imidazole，300mM NaCl，50mM NaH 2 PO 4 After dissolution, the pH was adjusted to 8.0 and distilled water was set to 1L.
5 XSDS-PAGE Loading buffer:1.25mL of 1M Tris-HCl (pH 6.8), 0.5g of SDS,0.025g of bromophenol blue and 2.5mL of glycerol, adding distilled water for dissolution, then fixing the volume to 5mL, split charging 1mL of each tube, and adding 25 mu L of beta-mercaptoethanol into each tube before use.
5 XTris-Glycine protein running buffer: 15.1g Tris,94g Glycine,5gSDS distilled water is fixed to a volume of 1L.
The main equipment comprises:
shaking table (Ningbo Saifu laboratory instruments Co., ltd.), biochemical incubator (Ningbo Jiangnan laboratory instruments Co., ltd.), high-speed low-temperature centrifuge (Dalong Xinghuang laboratory instruments (Beijing) Co., ltd.), ultrasonic breaker (Ningbo Xinzhi biotechnology Co., ltd.), water bath constant temperature oscillator (Shanghai Libang West instrument technology Co., ltd.), protein electrophoresis apparatus (Beijing junyi Oriental electrophoresis apparatus Co., ltd.), high performance liquid chromatography Agilent 1260 series combined with evaporative light scattering detector (Agilent technology Co., ltd.)
EXAMPLE two preparation of recombinant rhamnosidase
1. 50. Mu.L of E.coli competent cell BL21 (DE 3) and a plasmid containing the gene encoding the alpha-L-rhamnosidase of SEQ ID NO. 1 were added to pre-chilled culture tubes for transformation. After 20min on ice, heat shock was conducted for 45s at 42℃and then again placed on ice for 2min, 200. Mu.L of liquid LB medium was added to the culture tube, shaking culture was conducted for 1h at 37℃and then 60. Mu.L of the culture medium was spread on a solid LB plate containing 50. Mu.g/mL kanamycin resistance, and the culture was inverted overnight in a 37℃incubator.
The monoclonal was picked up and placed in kanamycin-resistant liquid LB medium and cultured overnight with shaking at 37 ℃.
2. 2mL of overnight culture broth was inoculated into 200mL of liquid LB medium containing 50. Mu.g/mL kanamycin resistance, and when the optical density (OD 600) of the broth reached 0.6 by 220rpm in a shaking table at 37℃the protein expression was induced by adding IPTG at a final concentration of 100. Mu.M, followed by culturing for 5 hours in a shaking table at 37℃at 200 rpm.
3. After the induction is completed, the bacterial cells are collected by centrifugation at 6000rpm for 10min, 10mL of Ni-NTA Lysis buffer is taken for resuspension, the collected bacterial cells are washed, the centrifugation at 6000rpm is carried out again for 10min, and then 15mL of Ni-NTA Lysis buffer is used for resuspension of the bacterial cells, and the bacterial cells are placed on ice for ultrasonic disruption. The crushing power is 30%, the total time is 30min, the ultrasonic time is 3s, and the interval time is 5s.
4. The cell lysate after ultrasonic disruption was centrifuged at 12000rpm at 4℃for 10min, and the supernatant was a crude enzyme solution, which was collected.
5. Purifying target protein by using a nickel column affinity chromatography, balancing a nickel column by using 10 Ni-NTA Lysis buffers with column volumes, slowly adding crude enzyme liquid into the nickel column after balancing, adding 20 Ni-NTA Washing buffer with column volumes to elute impurity proteins, eluting and collecting target protein by using Ni-NTA 250mM Elution buffer and Ni-NTA 350mM Elution buffer to obtain pure enzyme liquid, and obtaining recombinant rhamnosidase.
SDS-PAGE analysis and identification of the recombinant rhamnosidase expressed in E.coli (crude and pure enzyme solutions obtained in step 4 and step 5) are shown in FIG. 2, wherein M: protein markers; 1: crude enzyme solution; 2: purified recombinant rhamnosidase. The results show that the recombinant rhamnosidase of SEQ ID No. 1 has been obtained by recombinant expression.
SEQ ID NO. 1 is as follows:
MVHGLRIIDARVEFTVNPLGIDESKPRFSWILEHEERGQYQSAYRVIVSSSLENAVKGIGDVWDSGKVNS
RDQVIKYNGPPLSSFTKYYWRVKAWDSNGVEGDWSDVQWFETAVLKPEEWSGKWIGGGQLLRRSFRVEGS
VIEAKAYVTGLGYYELRINGERVGDRVLDPPWSEYDKTVYYSVYDVTNLVKSGENVIGLILGRGRYGPVS
PNRAQIPGLKYYDEPKASAMIRIRLSDGSVITINTDESWKCLVKGPILYDDIYNGYRYDARLEPYGWDKA
GFDDSNWVQCSVVKPPGGRLRSTAAVPGTKVKGTLKPREYYNPRPGVYVFDFGQNITGWVRLRVRGSSGV
EVKVRHSEVINSDGSLNVENIRGAEATDTYILSGRDVEVLEPRFTYHGFRYAEVTGYPGVPSIDDVEAVI
VQTDFESTGSIATSSKIINDIHRITWWSLRANLLNGIQTDCPQRDERMGWLGDAWLSSDSAVFNFNMVKY
YEKFIRDIIDSQRDDGSIPDTVPPYWNTYPADPAWGTALIYIPWLLYVHYGDVEILEEAYEAMKKWWSFL
NSRVKDNVLYFSKYGEWVPPGRVFSAEYCPPEILSTWILYRDTLTLAQIAKVLGRGEDASFFTKRAEEIR
DAFNRVFLTERGYYSKYTAPDGSVRMLGGSQTCNALPLYLDMVPGNRVNDIVKALAHNIEADWDRHLVVG
IFGAKYVPEVLVKYGYVDLAYRAVTQESYPGWGYMIKEGATTLWERWEKLTGAGMNSHNHHMFGSIDAWF
YRDLAGLMTLEPGFSRIMIKPNIPSELRYCSASLYTVRGLTSVEWSRVNDELVVTVTVPVNSTAEVHLPK
LGESTVVREGDKVLWSGGKVVEVSPGVLSVKDAGDRIVVEVGSGRFIFTIKTIN
example three properties of recombinant rhamnosidase
The recombinant rhamnosidase obtained in example two was subjected to detection of enzyme activity and its properties.
1. Enzyme activity determination method reaction system 500. Mu.L, 20. Mu.L 10mmol/L p-nitrophenyl alpha-L-rhamnoside (pNPR) adding 460. Mu.L 50mmol/L glycine-sodium hydroxide buffer (pH 10.0), incubating at 80 ℃ for 5min, adding 20. Mu.L enzyme solution (diluted to proper multiple) for reaction for 10min, and adding 1mol/L sodium carbonate solution 500. Mu.L for stopping reaction.
Absorbance was measured at 405 nm. The enzyme activity unit (U) is defined as: under the assay conditions, the amount of enzyme required to produce 1. Mu. Mol of p-nitrophenol per minute was 1 enzyme activity unit. The specific activity of the purified enzyme was 2974U/mg.
2. Determination of the optimum pH
Under different pH (2.0-12.0) conditions, respectively measuring enzyme activities at 80 ℃, and finding that the optimal reaction pH of the recombinant alpha-L-rhamnosidase is 10.0; the activity was maintained at 90% or more in the pH range of 5 to 10, and the results are shown in FIG. 3.
Determination of pH stability
After incubation for 2 hours at different pH (3.0-12.0) and at 80 ℃ and measurement of enzyme activity, the recombinant rhamnosidase can still maintain more than 95% of activity after being mixed with buffer solution with pH of 5-10 and incubated for 2 hours, and the recombinant alpha-L-rhamnosidase is alkaline rhamnosidase, has wider adaptation pH range and can maintain higher activity in an environment with stronger alkalinity, and the result is shown in figure 4.
4. Determination of optimum temperature
The enzyme activities were measured at intervals of 10℃in the range of 60-140℃over 100℃with an oil bath, and the optimum reaction temperature of the rhamnosidase was found to be 100℃and the rhamnosidase was found to be an alkaline thermophilic rhamnosidase, the results of which are shown in FIG. 5.
5. Temperature stability
The enzyme activity is measured in different time periods by incubating for 2 hours under different temperature (60 ℃ to 90 ℃), the alkaline thermophilic recombinant alpha-L-rhamnosidase has better temperature stability at 60 ℃ to 80 ℃, and the enzyme activity is 100% after 2 hours, and the result is shown in figure 6.
Example four transformation experiments of recombinant rhamnosidase on the respective substrates
Based on the above enzymatic properties, the recombinant rhamnosidase was selected to perform a transformation experiment on rutin, naringin, hesperidin and epimedin C under optimal conditions (80 ℃/ph=10), and the transformation rate was determined.
1. Transformation experiment conditions
The rutin conversion reaction system is 1L, and comprises 50mg of purified enzyme solution, 100g of rutin, 50mM of Glycine-NaOH pH=10 buffer solution, and 200r/min of water bath oscillation at 80 ℃.
The naringin and hesperidin conversion reaction system is 1L and comprises 50mg of purified enzyme solution, 100g of substrate and 50mM Na 2 HPO 4 -NaH 2 PO 4 In buffer with ph=8, shaking in a water bath at 80 ℃ for 200 r/min.
The epimedin C conversion reaction system is 1L, and comprises 50mg of purified enzyme solution, 100g of epimedin C and 50mM of Glycine-NaOH pH=10 buffer solution, and is oscillated in a water bath at 80 ℃ for 200 r/min.
2. Conversion product detection conditions
The rutin liquid phase detection condition chromatographic column adopts a C18 column, wherein the mobile phase A is 0.1% phosphoric acid water, the mobile phase B is acetonitrile, the detection wavelength is 270nm, the flow rate is 1ml/min, the sample injection amount is 5 mu L, the column temperature is 26 ℃, and the HPLC program is shown in Table 1.
TABLE 1 rutin liquid phase detection Condition HPLC procedure
Time (min) Mobile phase a (%) Mobile phase B%％)
0 75 25
16 73 27
23 64 36
25 75 25
30 75 25
The naringin liquid phase detection condition chromatographic column adopts a C18 column, the mobile phase is water and methanol, the ratio is 71:29, the detection wavelength is 283nm, the flow rate is 1ml/min, the sample injection amount is 5 mu L, and the column temperature is 26 ℃.
The hesperidin liquid phase detection condition chromatographic column adopts a C18 column, the mobile phase is water and methanol, and the proportion is 50:50, detection wavelength 280nm, flow rate 1ml/min, sample injection amount 5 μl, column temperature 26 ℃.
The icariin liquid phase detection condition chromatographic column adopts a C18 column, the mobile phase is acetonitrile and water, and the proportion is 26:74, the detection wavelength is 270nm, the flow rate is 1ml/min, the sample injection amount is 5 mu L, and the column temperature is 26 ℃.
3. Conversion results
Carrying out HPLC detection on the converted rutin, reacting for 4 hours, wherein the conversion rate reaches more than 98.5%, and the result is shown in figure 7;
naringin is converted and is subjected to HPLC detection, the reaction is carried out for 6 hours, the conversion rate is more than 98.5%, and the result is shown in figure 8;
performing HPLC detection on the converted hesperidin, reacting for 8 hours, wherein the conversion rate is more than 98.5%, and the result is shown in figure 9;
the epimedin C is converted into icariin and is subjected to HPLC detection, the reaction is carried out for 6 hours, the conversion rate is more than 98.5%, and the result is shown in figure 10.
In conclusion, the alkaline thermophilic rhamnosidase can biologically convert rutin, naringin, hesperidin, epimedin C and other flavonoid compounds at high temperature and strong alkali, and the conversion rate can reach more than 98.5%.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Sequence listing
<110> Beijing Tuo Bio-technology Co., ltd
<120> application of alkaline thermophilic rhamnosidase in preparing active bioflavonoids
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 894
<212> PRT
<213> artificial sequence
<400> 1
Met Val His Gly Leu Arg Ile Ile Asp Ala Arg Val Glu Phe Thr Val
1 5 10 15
Asn Pro Leu Gly Ile Asp Glu Ser Lys Pro Arg Phe Ser Trp Ile Leu
20 25 30
Glu His Glu Glu Arg Gly Gln Tyr Gln Ser Ala Tyr Arg Val Ile Val
35 40 45
Ser Ser Ser Leu Glu Asn Ala Val Lys Gly Ile Gly Asp Val Trp Asp
50 55 60
Ser Gly Lys Val Asn Ser Arg Asp Gln Val Ile Lys Tyr Asn Gly Pro
65 70 75 80
Pro Leu Ser Ser Phe Thr Lys Tyr Tyr Trp Arg Val Lys Ala Trp Asp
85 90 95
Ser Asn Gly Val Glu Gly Asp Trp Ser Asp Val Gln Trp Phe Glu Thr
100 105 110
Ala Val Leu Lys Pro Glu Glu Trp Ser Gly Lys Trp Ile Gly Gly Gly
115 120 125
Gln Leu Leu Arg Arg Ser Phe Arg Val Glu Gly Ser Val Ile Glu Ala
130 135 140
Lys Ala Tyr Val Thr Gly Leu Gly Tyr Tyr Glu Leu Arg Ile Asn Gly
145 150 155 160
Glu Arg Val Gly Asp Arg Val Leu Asp Pro Pro Trp Ser Glu Tyr Asp
165 170 175
Lys Thr Val Tyr Tyr Ser Val Tyr Asp Val Thr Asn Leu Val Lys Ser
180 185 190
Gly Glu Asn Val Ile Gly Leu Ile Leu Gly Arg Gly Arg Tyr Gly Pro
195 200 205
Val Ser Pro Asn Arg Ala Gln Ile Pro Gly Leu Lys Tyr Tyr Asp Glu
210 215 220
Pro Lys Ala Ser Ala Met Ile Arg Ile Arg Leu Ser Asp Gly Ser Val
225 230 235 240
Ile Thr Ile Asn Thr Asp Glu Ser Trp Lys Cys Leu Val Lys Gly Pro
245 250 255
Ile Leu Tyr Asp Asp Ile Tyr Asn Gly Tyr Arg Tyr Asp Ala Arg Leu
260 265 270
Glu Pro Tyr Gly Trp Asp Lys Ala Gly Phe Asp Asp Ser Asn Trp Val
275 280 285
Gln Cys Ser Val Val Lys Pro Pro Gly Gly Arg Leu Arg Ser Thr Ala
290 295 300
Ala Val Pro Gly Thr Lys Val Lys Gly Thr Leu Lys Pro Arg Glu Tyr
305 310 315 320
Tyr Asn Pro Arg Pro Gly Val Tyr Val Phe Asp Phe Gly Gln Asn Ile
325 330 335
Thr Gly Trp Val Arg Leu Arg Val Arg Gly Ser Ser Gly Val Glu Val
340 345 350
Lys Val Arg His Ser Glu Val Ile Asn Ser Asp Gly Ser Leu Asn Val
355 360 365
Glu Asn Ile Arg Gly Ala Glu Ala Thr Asp Thr Tyr Ile Leu Ser Gly
370 375 380
Arg Asp Val Glu Val Leu Glu Pro Arg Phe Thr Tyr His Gly Phe Arg
385 390 395 400
Tyr Ala Glu Val Thr Gly Tyr Pro Gly Val Pro Ser Ile Asp Asp Val
405 410 415
Glu Ala Val Ile Val Gln Thr Asp Phe Glu Ser Thr Gly Ser Ile Ala
420 425 430
Thr Ser Ser Lys Ile Ile Asn Asp Ile His Arg Ile Thr Trp Trp Ser
435 440 445
Leu Arg Ala Asn Leu Leu Asn Gly Ile Gln Thr Asp Cys Pro Gln Arg
450 455 460
Asp Glu Arg Met Gly Trp Leu Gly Asp Ala Trp Leu Ser Ser Asp Ser
465 470 475 480
Ala Val Phe Asn Phe Asn Met Val Lys Tyr Tyr Glu Lys Phe Ile Arg
485 490 495
Asp Ile Ile Asp Ser Gln Arg Asp Asp Gly Ser Ile Pro Asp Thr Val
500 505 510
Pro Pro Tyr Trp Asn Thr Tyr Pro Ala Asp Pro Ala Trp Gly Thr Ala
515 520 525
Leu Ile Tyr Ile Pro Trp Leu Leu Tyr Val His Tyr Gly Asp Val Glu
530 535 540
Ile Leu Glu Glu Ala Tyr Glu Ala Met Lys Lys Trp Trp Ser Phe Leu
545 550 555 560
Asn Ser Arg Val Lys Asp Asn Val Leu Tyr Phe Ser Lys Tyr Gly Glu
565 570 575
Trp Val Pro Pro Gly Arg Val Phe Ser Ala Glu Tyr Cys Pro Pro Glu
580 585 590
Ile Leu Ser Thr Trp Ile Leu Tyr Arg Asp Thr Leu Thr Leu Ala Gln
595 600 605
Ile Ala Lys Val Leu Gly Arg Gly Glu Asp Ala Ser Phe Phe Thr Lys
610 615 620
Arg Ala Glu Glu Ile Arg Asp Ala Phe Asn Arg Val Phe Leu Thr Glu Arg
625 630 635 640
Gly Tyr Tyr Ser Lys Tyr Thr Ala Pro Asp Gly Ser Val Arg Met Leu
645 650 655
Gly Gly Ser Gln Thr Cys Asn Ala Leu Pro Leu Tyr Leu Asp Met Val
660 665 670
Pro Gly Asn Arg Val Asn Asp Ile Val Lys Ala Leu Ala His Asn Ile
675 680 685
Glu Ala Asp Trp Asp Arg His Leu Val Val Gly Ile Phe Gly Ala Lys
690 695 700
Tyr Val Pro Glu Val Leu Val Lys Tyr Gly Tyr Val Asp Leu Ala Tyr
705 710 715 720
Arg Ala Val Thr Gln Glu Ser Tyr Pro Gly Trp Gly Tyr Met Ile Lys
725 730 735
Glu Gly Ala Thr Thr Leu Trp Glu Arg Trp Glu Lys Leu Thr Gly Ala
740 745 750
Gly Met Asn Ser His Asn His His Met Phe Gly Ser Ile Asp Ala Trp
755 760 765
Phe Tyr Arg Asp Leu Ala Gly Leu Met Thr Leu Glu Pro Gly Phe Ser
770 775 780
Arg Ile Met Ile Lys Pro Asn Ile Pro Ser Glu Leu Arg Tyr Cys Ser
785 790 795 800
Ala Ser Leu Tyr Thr Val Arg Gly Leu Thr Ser Val Glu Trp Ser Arg
805 810 815
Val Asn Asp Glu Leu Val Val Thr Val Thr Val Pro Val Asn Ser Thr
820 825 830
Ala Glu Val His Leu Pro Lys Leu Gly Glu Ser Thr Val Val Arg Glu
835 840 845
Gly Asp Lys Val Leu Trp Ser Gly Gly Lys Val Val Glu Val Ser Pro
850 855 860
Gly Val Leu Ser Val Lys Asp Ala Gly Asp Arg Ile Val Val Glu Val
865 870 875 880
Gly Ser Gly Arg Phe Ile Phe Thr Ile Lys Thr Ile Asn
885 890

Claims (4)

1. A method for bioconverting flavonoid compounds with high conversion rate, which is characterized in that the bioconversion is carried out under the conditions of high temperature and different pH values, the bioconversion of flavonoid compounds is carried out under the action of alkaline thermophilic rhamnosidase, and the amino acid sequence of the alkaline thermophilic rhamnosidase is shown as SEQ ID NO:1, wherein the flavonoid compound is selected from one or more of rutin, naringin, hesperidin, neohesperidin or epimedin C, the high temperature condition is 80-100 ℃, and the different pH conditions are pH8-10.
2. The method of claim 1, wherein the high temperature condition is 80 ℃ or 100 ℃.
3. The application of alkaline thermophilic rhamnosidase in bioconversion of flavonoid compounds under high temperature conditions and different pH conditions is characterized in that the amino acid sequence of the alkaline thermophilic rhamnosidase is shown in SEQ ID NO:1, wherein the flavonoid compound is selected from one or more of rutin, naringin, hesperidin, neohesperidin or epimedin C, the high temperature condition is 80-100 ℃, and the different pH conditions are pH8-10.
4. Use according to claim 3, characterized in that the high temperature condition is 80 ℃ or 100 ℃.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104312996A (en) * 2014-09-26 2015-01-28 南京林业大学 Alpha-L-rhamnosidase Rha1 as well as expressed gene and application of alpha-L-rhamnosidase Rha1
CN104762281A (en) * 2015-03-09 2015-07-08 南京林业大学 Alpha-L-rhamnosidase and preparing method and applications thereof
CN108467858A (en) * 2018-02-05 2018-08-31 南京林业大学 A kind of alpha-L-Rhamnosidase and its application
CN112210548A (en) * 2020-09-07 2021-01-12 佛山市汇腾生物技术有限公司 Pichia pastoris for expressing alpha-L-rhamnosidase and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104312996A (en) * 2014-09-26 2015-01-28 南京林业大学 Alpha-L-rhamnosidase Rha1 as well as expressed gene and application of alpha-L-rhamnosidase Rha1
CN104762281A (en) * 2015-03-09 2015-07-08 南京林业大学 Alpha-L-rhamnosidase and preparing method and applications thereof
CN108467858A (en) * 2018-02-05 2018-08-31 南京林业大学 A kind of alpha-L-Rhamnosidase and its application
CN112210548A (en) * 2020-09-07 2021-01-12 佛山市汇腾生物技术有限公司 Pichia pastoris for expressing alpha-L-rhamnosidase and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
alpha-L-rhamnosidase [Caldivirga maquilingensis];无;《GENBANK》;序列部分 *
无.alpha-L-rhamnosidase [Caldivirga maquilingensis].《GENBANK》.2019,序列部分. *

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